Force-Induced Shuttling of Rotaxanes Controls Fluorescence Resonance Energy Transfer in Polymer Hydrogels

The molecular shuttling function of rotaxanes can be exploited to design mechanoresponsive reporter molecules. Here, we report a new approach to such rotaxane-based mechanophores, in which the fluorescence resonance energy transfer (FRET) between a donor–acceptor pair is mechanically controlled. A cyclic molecule containing a green-light-emitting FRET donor connected to a red-light-emitting FRET acceptor was threaded onto an axle equipped with a quencher at its center and two stoppers in the peripheral positions. In the force-free state, the green emitter is located near the quencher so that charge transfer interactions or photo-induced electron transfer between the two moieties suppress green emission and prevent the FRET from the green to the red emitter. The mechanophore was covalently incorporated into a linear polyurethane-urea (PUU), and stretchable hydrogels were prepared by swelling this polymer with water. Upon deformation of the PUU hydrogels and under an excitation light that selectively excites the donor, the intensity of the red fluorescence increases, as a result of a force-induced separation of the green emitter from the quencher, which enables the FRET. The switching contrast is much more pronounced in the gels than in dry films, which is due to increased molecular mobility and hydrophobic effects in the hydrogel, which both promote the formation of inclusion complexes between the ring containing the green emitter and the quencher.


Calculation of Spectral Overlap Integral and Förster distance
Using equations (1) and (2), the spectral overlap integral (J(λ)) between the donor fluorescence and the acceptor absorption, and the Förster distance (R0) of the donor/acceptor pair were determined: S4 (1) Here, f(λ) is the normalized fluorescence intensity of the 9,10-bis(phenylethynyl)anthracene donor, ε(λ) is the extinction coefficient of the π-extended BODIPY acceptor, N is Avogadro's number, κ 2 is the orientation factor of 2/3, φD is the fluorescence quantum yield of the donor (0.91 S1 ), n is the refractive index of the 1:4, v:v THF/methanol solution (estimated to be 1.345), and the constants preceding κ have an aggregate value of 8.79 × 10 -11 M cm nm 2 .
Using these values, J(λ) and R0 were calculated to be 5.581 × 10 15 M -1 cm -1 nm 4 and 67.1 Å, respectively. The energy transfer efficiency (E) is described by equation (3), where r is the distance between the donor and acceptor molecules. Using the value of the Förster distance reported above, the energy transfer efficiency should be higher than 99% when r is below 31.2 Å.   Figure S7. DMA traces of dried RotAnBP-PUU films. The graph shows data obtained from three different specimens.

Mechanical Properties of Dried RotAnBP-PUU Films and RotAnBP-PUU Hydrogels
The tests were conducted under N2 at a heating rate of 3 °C/min, a frequency of 1 Hz, and an amplitude of 15 μm. S11 Figure S8. Strain-stress curves of (a) dried RotAnBP-PUU films and (b) RotAnBP-PUU hydrogels. Each graph shows data obtained from three different specimens. The experiments were conducted with a strain rate of 60 mm/min at r.t.  Figure S8 and represent averages of 3 measurements ± standard derivation. b) The Young's moduli were derived from the slopes of the strain-stress curves in the strain regime between 1.5-2%.  Figure S11. Pictures of (a) RotAnBP-PUU hydrogel and (b) dried RotAnBP-PUU films during uniaxial deformation test. In each strained state, the top picture displays the fluorescence of the hydrogel; these images were taken in the dark upon excitation at 490 nm, and a long pass filter cutting below 550 nm was placed in front of the camera. The bottom picture shown for each strain was taken under ambient illumination. All images were taken under the same ambient conditions. Figure S12. Fluorescence spectra of RotAnBP-PUU hydrogel recorded upon relaxation from the maximum strain of 1000% in the first cycle. The fluorescence spectra were recorded at r.t. with λex = 490 nm. Figure S13. Fluorescence spectra of RotAnBP-PUU hydrogel upon stretching the samples to the strains indicated in the first cycle. The fluorescence spectra were recorded at r.t. with λex = 590 nm.

Examination of the Penetration of the Red Fluorescence Emitted by RotAnBP-PUU Hydrogels Preparation of RotAnBP-PUU Hydrogels Based on a Second Batch of RotAnBP-PUU.
In this experiment, RotAnBP-PUU hydrogels were prepared using a second batch of RotAnBP-PUU that was synthesized according to the procedure and reaction scheme described above (Scheme S5). The mechanical response of the newly prepared hydrogel ( Figure S14) is similar to that of the hydrogels used in other experiments ( Figure S8). Figure S14. Stress-strain curve of a RotAnBP-PUU hydrogel based on a second batch of RotAnBP-PUU. The experiment was conducted with a strain rate of 60 mm/min at r.t.

Reference Polyurethane Films in which a Rotaxane Mechanophore Showing On/Off Switching of Green Emission Was Incorporated.
For reference purposes, a polyurethane that we reported as Rot2PU in a previous paper S5 and which contained the green-light emitting rotaxane Rot 2 was employed. The molecular structures of Rot2 and Rot2PU are shown in Figure S15.

Preparation of Red PU Films without Any Mechanophores.
The red-colored PU films were prepared by physically doping sudan III ( Figure S16a and S16b) into the reference polyurethane without any mechanophores. Thus, PU (300 mg, Mn = 121 kg/mol, PDI = 2.04) synthesized according to the method that we previously reported S1 and 10 mg of sudan III were dissolved in THF (5 mL) and the solution was divided between two square poly(tetrafluoroethylene) molds (51 × 51 × 5.0 mm). The molds were placed under an inverted funnel to control the evaporation of the solvent. The solvent was evaporated over the course of 4 h under ambient conditions and the resulting films were further dried in vacuo at r.t. for 12 h. The red films thus obtained were smooth and opaque with a thickness of 60-80 μm. A characteristic stress-strain curve of the films thus obtained is shown in Figure S16c.

Changes in Fluorescence Spectra of Hydrogels and Films Overlaid with the Red Films upon Stretching.
Tensile tests were performed for samples of RotAnBP-PUU hydrogel or Rot2PU film overlaid without and with the red films ( Figure S17). While stretching tests, the excitation light was irradiated to the center of RotAnBP-PUU hydrogel or Rot2PU film from one optical fiber connected to the light source, and the fluorescence of the RotAnBP-PUU hydrogel or Rot2PU film passing through the red PU film was monitored by the other optical fiber connected to the detector.

Supporting Movies
Movies S1 and S2. Representative movies of the mechanoresponsive luminescent behavior upon cyclic stretching of a RotAnBP-PUU hydrogel and a dried RotAnBP-PUU film. The movies were taken in the dark under excitation at 490 nm and a long pass filter cutting below 550 nm was placed in front of the camera.